In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor

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Abstract

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.

Introduction

The paracrine hormone GLP-1 is secreted by the duodenum in response to high glucose levels, which subsequently triggers the release of insulin from pancreatic cells [1], [2]. The ability of GLP-1 to induce insulin secretion in dependence on high glucose levels, renders this component potentially useful in the treatment of non-insulin dependent diabetus mellitus (NIDDM) [3], [4]. However, the peptide nature of GLP-1 prevents its direct application due to its short half-life in vivo [4]. A rational design of new agonists will therefore greatly benefit from structural knowledge of the interaction between GLP-1 and its receptor.

The receptor protein for GLP1 was first cloned by Thorens [5] in 1992; it belongs to class B of G-protein coupled receptors. The protein has a large N-terminal extracellular domain that was shown to be involved in the ligand binding [6], [7], [8]. As for other peptide–hormone receptors such as receptors for parathyroid hormone (PTH) [9], vasoactive intestinal peptide (VIP) [10], calcitonin [11] or pituitary adenylate cyclase activating peptide (PACAP) [12], the N-terminal domain contains six conserved cysteine residues, which are assumed to form three disulfide bonds. It was shown for the PTH-receptor [13], [14], [15] and the VIP-receptor [16] that intact disulfide bonds are essential for functionality.

Unfortunately, receptors of this class are not yet available in amounts that will allow their structural analysis. To date, only the receptors for vasoactive intestinal peptide (VIP) [17] and pituitary adenylate cyclase activating peptide (PACAP) [18] have been purified in the sub-milligram range from overexpressing eucaryotic cell lines.

As the extracellular domain of the GLP-1 receptor can specifically bind its peptide [19], we set out to generate large amounts of that portion of the molecule for functional and structural analysis.

Section snippets

Construction of expression plasmid p(nGLP1R)

A gene fragment, coding for the extracellular part of the human GLP-1 receptor, nGLP1R, was amplified by PCR from a cDNA encoding the human GLP-1 receptor using primers 5′-AAAGAGCTCGCCGGCCCCCGCCCC-3′ and 5′-TTTAAGCTTTTATTTGCGTTTGCGT-TTGCGTTTGCGACCGTAGAGGAACAGGAGCTG -3′. The fragment was inserted into pQE30 (Qiagen) using SphI and HindIII restriction sites, and in a second step the codons for four amino acids Ala, Cys, Glu, and Leu between the hexahistidine tag and the coding region of the

Expression of recombinant protein

A fragment encoding the N-terminal 125 amino acids of the GLP-1 receptor without the putative signal sequence was amplified by PCR from a plasmid containing the entire coding sequence of the human GLP-1 receptor [28] and inserted into a pQE30-vector. The downstream primer contained a coding region for a stretch of eight consecutive positively charged residues to generate a polyionic tag. Vector insertion led to the addition of a further 16 amino acids at the N-terminus of the receptor fragment

Discussion

The receptor for GLP-1 belongs to the class B of GPCRs that bind to oligopeptidic ligands [37]. Due to the poor accessibility of all these receptors from natural sources only two of them, the receptor for vasointestinal peptide and that for pituitary adenylate cyclase activating peptide, have been isolated so far. Still, these quantities do not allow structural characterization of the receptors. We therefore decided to utilize E. coli as a high-level expression system and to refold the protein

Acknowledgements

We thank Dr Andreas Wilmen for the cDNA of the human GLP-1 receptor, Dr Karl Peter Rücknagel for N-terminal sequencing of the expressed receptor fragment, Achim Gärtner for N-terminal sequencing of the peptide fragments used for disulfide bond analyses, Bettina Foelting for conduction of DSC scans, Dr Hauke Lilie, Bjoern Schott, and Dr Christopher Rensing for critically reading the manuscript.

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